Bioactivity of Cereal- and Legume-Based Macaroni Pasta Volatiles to Adult Sitophilus granarius (L.)

Simple Summary Pasta factories can be infested by insects. By following the odour of cereal-based pasta, insects can enter packages of commercial products. The aim of this work was to compare the bioactivity of volatiles produced by cereal- and legume-based macaroni pasta on adults of granary weevil, Sitophilus granarius, in multi-choice behavioural bioassays. Tests were performed with ten commercially available Italian macaroni pastas made from six different cereals or four different legumes. Granary weevil adults were more attracted to cereal-based pastas than legume-based pastas, but the differences in attractiveness were not always significant. Gas chromatography-mass spectrometry analysis of head-space solid-phase microextraction collections from the different pasta samples highlighted marked qualitative and quantitative differences, with aliphatic aldehydes and aliphatic alcohols being the most abundant volatile components of cereal- and legume-pastas, respectively. Moreover, the results of the two-choice behavioural bioassays suggested that the low level of attraction to legume pasta is mainly due to the lack of attractant stimuli other than emission of repellent compounds. Abstract The attractiveness of ten commercially available Italian macaroni pastas made from different cereals [Triticum durum; Triticum durum (whole wheat); Triticum dicoccum; mixture of five cereals; Triticum turgidum; Triticum turanicum] or legumes (Cicer arietinum; Lens culinaris; Pisum sativum; Vicia faba) to Sitophilus granarius, was compared. S. granarius adults were more attracted to cereal pastas than legume pastas, but the differences in attractiveness were not always significant. Consistent with the results of behavioural bioassays, the mortality of adults over 20 days exposed to pasta samples was 100% with the legume pasta samples and only 8% with the T. turanicum pasta. GC-MS analysis of HS-SPME extracts from the different pasta samples highlighted marked qualitative and quantitative differences, with aliphatic aldehydes and aliphatic alcohols being the most abundant volatile components of cereal- and legume-pastas, respectively. In two-choice behavioural bioassays, insect attraction to a 1:1 combination of T. turanicum and C. arietinum pastas (80%) was even higher than that observed in T. turanicum pasta alone (64%) and in C. arietinum pasta alone (20%). This strongly suggested that the low attractiveness of legume pasta is mainly due to the lack of attractant stimuli rather than emission of repellent compounds.

In particular, dried cereal pasta can be infested with Sitophilus spp. during shipment in trucks, railcars and ships, as well as during retail storage, or even in the consumer's home [6,[9][10][11][12]. Following the odour of cereal-based pasta, adult weevils (as penetrator) can enter packages of commercial products enlarging the air vent micro-holes, through the openings made by them, or through the existing openings created by poor seals or mechanical damage [6,[11][12][13][14][15].
In recent years, consumers are requiring new types of pasta with healthy nutritional characteristics, as a good source of plant-based protein and fibre, certified gluten free and non-GMO. For these reasons, the food industry has produced and marketed pasta made with flours derived from legumes. The legume-based pasta production process is very similar to that of wheat-based pasta. The dried legumes are milled and then run through fine-mesh sieves until only a fine-textured flour remains. Water is mixed into the flour, other ingredients are sometimes added, and then the dough is kneaded and finally extruded through dies into various shapes.
To our knowledge, there is no published scientific literature that considers Sitophilus granarius (L.) infestations in dried pasta made with legume flour. However, there are some studies that consider the relationship between S. granarius adults and legume seeds [25,26].
The granary weevil, S. granarius, is distributed throughout the temperate regions of the world and is a frequent pest of wheat, also attacking barley, maize, sorghum, rice, and other cereal grains. Larval stages feed inside the kernels, leaving only the hulls. They sometimes infest sunflower seeds, dried beans, chickpeas, peanuts, fava beans, acorns, chestnuts, pasta products, and ornamental dried corn [9,27].
It is known that legume seeds contain a wide range of allelochemicals with toxic and deterrent effects against insect pests [28,29]. An admixture of yellow split-peas (Pisum sativum L.) with wheat resulted in a marked reduction in the survival and reproduction rate of Sitophilus oryzae (L.) [30,31].
Concentrations as low as 0.01% pea protein were shown to cause adult mortality and reduced reproduction for several stored-product insect pests [32,33]. The repellence of pea seed fractions to stored-product insect pests has been demonstrated in multiple-choice tests, in which wheat kernels were dusted with fractions rich in either protein, fibre or starch. This result is probably due to either the olfactory or gustatory effects of the pea fractions [25,26]. However, to the best of our knowledge, semiochemical interactions between stored-product pests and legume-derived products remain little investigated.
The main purpose of this work was to compare the bioactivity of volatiles produced by cereal-and legume-based macaroni pasta against adults of S. granarius in multichoice behavioural bioassays. Moreover, head-space solid-phase microextraction (HS-SPME) extracts from the different types of pasta tested in bioassays were analysed by gas-chromatography coupled with mass-spectrometry (GC-MS) to highlight differences between their odour blends.

Insects
A wild S. granarius population found and reared on barley with no history of exposure to insecticides was maintained in a climatic chamber at 28 ± 2 • C and 70 ± 5% RH, with an L12:D12 photoperiod. Unsexed 1 to 2-week-old adults were used in behavioural bioassays and susceptibility tests.
The nutritional information (average values) for 100 g of each macaroni pasta is reported in Table 1.

Multiple-Choice Behavioural Bioassays
The attractiveness of different macaroni pastas to adult granary weevil adults was compared using a circular olfactometer arena (100 cm diameter, 50 cm height) similar to that described in previous studies [5].
Modified Petri dishes (9 cm diameter), each baited with a sample (25 g) of different macaroni pastas, were equally spaced along the edge of the arena, and 100 S. granarius adults were released at its centre. To prevent insect escape, Teflon paint was applied to the arena walls. The number of "trapped" insects was counted 1 day and 7 days after their introduction to the arena. After each experiment, baits were renewed and the positions of the different Petri dishes were randomly assigned. Tests were carried out in the dark at 28 ± 2 • C and 70 ± 5% RH. For each experiment, six replicates were performed.

Two-Choice Behavioural Bioassays
The attractiveness of Khorasan pasta (F) and chickpea pasta (H) was compared using the circular olfactometer arena, the modified Petri dishes and the methodology described above. The numbers of trapped and untrapped (free in the arena) insects were checked 1 day after their introduction to the arena. The following pairwise comparisons were performed (First vs. Second choice): 25 g of Triticum turanicum vs. control (empty modified Petri dish); 25 g of Cicer arietinum vs. control; mixed 25 g of Triticum turanicum and 25 g of Cicer arietinum vs. control; mixed 50% Triticum turanicum and 50% Cicer arietinum vs. mixed 75% Triticum turanicum and 25% Cicer arietinum; mixed 50% Triticum turanicum and 50% Cicer arietinum vs. mixed 25% Triticum turanicum and 75% Cicer arietinum; and 100% Triticum turanicum vs. 100% Cicer arietinum. For each experiment, four replicates were performed (see Table 2). In each trial, a response index (RI) was calculated using RI = [(T − C)/Tot] × 100, where T is the number of insects responding to the first choice, C is the number responding to the second choice, and Tot is the total number of insects released [13].

Susceptibility of Pasta Samples
For each type of pasta tested, 25 g samples were placed in wide-necked vials (100 mL volume) and infested with 30 unsexed 1 to 2-week-old granary weevil adults. To ensure air exchange, a series of small holes were punched in the vial screw caps. Vials were maintained in the dark at 28 ± 2 • C and 70 ± 5% RH. Granary weevil mortality was recorded 20 days after the start of the experiment, and emergence of new adults (F1) was checked every 2 days for 8 consecutive weeks. For each experiment, four replicates were performed.

Extraction of Pasta Volatiles
To identify and quantify the volatile compounds emitted by the different types of pasta tested in the behavioural bioassays, the static head-space solid-phase micro-extraction (HS-SPME) technique was used according to Beleggia et al. (2009) and Germinara et al. (2019) [34,35]. Pastas were stored in their unopened packages at room temperature until use. Before analysis, 10 g of each pasta sample was ground in an electric mill for 20 s at 6000 rpm (Waring ® laboratory blenders, Fisher scientific, Göteborg, Sweden) and placed in a 20 mL headspace vial (Supelco Co., Bellefonte, PA, USA), sealed with a PTFE/silicon septum (Supelco Co., Bellefonte, PA, USA) for analysis. The vial was then conditioned at 50 • C for 30 min in a water bath prior to SPME headspace sampling.
Extraction was performed using SPME fibres (Supelco Co., Bellefonte, PA, USA) coated with either 50/30 µm of divinylbenzene-carboxen-polydimethylsiloxane (DVB-CAR-PDMS). The fibres were conditioned before use by heating them in the injection port of the GC system, according to the manufacturer's recommendations, at 270 • C for 1 h. Then, the SPME needle was introduced through the septum, and the fibre was exposed in the vial to the headspace of the pasta sample for 90 min. A temperature of 50 • C was maintained during headspace sampling. After the extraction time, the fibre was recovered and transferred to the injection port of the GC, where the compounds were thermally desorbed at 250 • C for 4 min. A fibre cleaning step of 10 min at the conditioning temperature with the split valve opened was performed in the GC injector after every chromatographic run to remove any absorbed residue. Before the acquisitions, a blank test was performed under the same experimental conditions to check for possible impurities. Each sampling was performed in triplicate.

Gas Chromatography-Mass Spectrometry (GC-MS)
GC-MS analyses were performed using an Agilent 7890B series gas chromatograph (Agilent Technologies, Milan, Italy) coupled with an Agilent 5977A mass selective detector (MSD) equipped with an HP-5MS capillary column (30 m × 0.25 mm ID, 0.5 µm film thickness, J&W Scientific Inc., Folsom, CA, USA). The desorption step was carried out in the splitless mode (4 min) with a programmed temperature from 60 • C to 250 • C at 5 • C/min, with a final holding time of 15 min. Spectra were recorded in the electron impact mode (ionization energy, 70 eV) in a range of 15-550 amu at 2.9 scans/s. The identification of volatile compounds was achieved by comparing mass spectra with those of the data system library (NIST08, p > 90%), and, wherever possible, by comparing retention times (R.T.) and mass spectra with those of commercially available standards. Moreover, a mixture of a continuous series of straight-chain hydrocarbons, C5-C40 (Alkane Standard Solution C6-C40, Sigma Aldrich, Milan, Italy), was injected into an HP-5MS column under the same conditions previously described for the pasta samples to obtain the linear retention indices (RIs) [36]. Component relative percentages were calculated based on GC peak areas. Each extract was analysed in triplicate.

Data Analysis
For the multiple-choice behavioural bioassays, the numbers of insects found in the different dishes were subjected to Friedman two-way ANOVA by ranks. In the case of significance (p < 0.05), the Wilcoxon signed ranks test was used for separation of means.
For the two-choice behavioural bioassays, Student's t-test was used to compare the mean numbers of insects found in the two choices.
The data on susceptibility of pasta samples were submitted to one-way analysis of variance (ANOVA). Means were separated using the Tukey-Kramer honest significant difference (HSD) test at the 0.05 significance level [37].

Multiple-Choice Behavioural Bioassays
Olfactory responses of granary weevil adults to different pasta samples in multiplechoice behavioural bioassays are reported in Figures 1 and 2. Significant differences in adults captured by the Petri dishes containing different macaroni pastas were recorded 1 day (χ 2 = 35.029, df = 9, p < 0.001) and 7 days (χ 2 = 35.553, df = 9, p < 0.001) after the start of the experiment.
(Wilcoxon test, p < 0.05) than those attracted to the durum wheat, spelt and various legume-based pastas. The latter were the weakest attractants, without significant differences among them (Wilcoxon test, p > 0.05).   (Wilcoxon test, p < 0.05) than those attracted to the durum wheat, spelt and various legume-based pastas. The latter were the weakest attractants, without significant differences among them (Wilcoxon test, p > 0.05).

Two-Choice Behavioural Bioassays
The results of the two-choice behavioural bioassays are reported in Table 2. When individually compared with an empty Petri dish, Khorasan, chickpea, and a 1:1 mixture of both pastas attracted 64%, 20%, and 80% of test insects, respectively. In total, 73.5% of insects chose the Khorasan pasta when presented with Khorasan pasta and chickpea pasta, whereas a preferential orientation of adult insects was not observed when making a choice between mixtures of Khorasan and chickpea pastas in different proportions.

Susceptibility of Pasta Samples
The mean numbers of live and dead S. granarius adults 20 days after exposure to different pasta samples in wide-necked vials are reported in Table 3. According to our results, significant differences were recorded in weevil mortality (F = 32.869; df = 9, 30; p < 0.01). A 100% mortality rate of adult insects was recorded in all legume-based pastas, significantly higher than those recorded for durum wheat pasta (70.00%), five cereal pasta (68.33%), and Khorasan pasta (27.50%). After the incubation period, new adults (F1 progeny) emerged from the Khorasan pasta (ten samples) and from the durum whole wheat pasta (one sample). No adults emerged from the other types of macaroni pasta.

Characterisation of Pasta Volatiles
HS-SPME/GC-MS was used to detect the main components in the odour profile of the different pasta samples. The percentages of specific compounds, expressed as relative abundance, are reported in Table 4. Across all ten pastas, a total of 50 volatile compounds in the chemical classes of alcohols, aldehydes, ketones, esters, lactones, terpenes, hydrocarbons, furans, and other compounds were detected.  A total of 27, 34, 40, 34, 33, and 33 volatile compounds were respectively identified in the head-space of samples A, B, C, D, E, and F, obtained from different cereals, whereas 42, 43, 30, and 40 compounds were identified from the G, H, I, and J legume-based pasta samples.
In cereal-based pasta samples, aldehydes were the most represented chemical class, both in terms of the number of compounds (10-13) and relative abundance (40.14-61.76%), followed by alcohols, aromatics, furans, and hydrocarbons. In the head-space volatile fraction of legume-based pasta samples, alcohols were the most numerous (11)(12) and abundant (53.8-61.15) among different chemical classes, followed by aldehydes, aromatics, furans, and lactones.

Discussion
The results of multiple-choice behavioural bioassays showed that S. granarius adults are able to selectively respond to odours of different types of Italian cereal-and legumebased pastas. In fact, in both the day one and day seven tests, cereal-based pastas were overall more attractive than legume-based ones. Among all pasta samples tested, those made from T. turanicum (Khorasan) and five cereals (wheat, spelt, barley, maize, and rye) were the most attractive. Whereas, those from whole wheat T. durum, T. dicoccum, and particularly from different legumes, were the weakest attractants.
To investigate the nature of the low attractiveness of legume-based pastas to granary weevil adults, two-choice behavioural bioassays were performed. In these experiments, pastas obtained from T. turanicum (Khorasan pasta) and C. arietinum (chickpea pasta) confirmed their respective high and low attractiveness to adult insects. Whereas, a 1:1 mixture of the two pastas elicited a significant insect attraction, comparable to that of T. turanicum pasta. This strongly suggests that the low attractiveness of legume-based pastas to granary weevils mainly depends on the lack of attractive odour stimuli rather than the presence of repellent volatile compounds. This hypothesis was further confirmed by pairwise comparisons of different mixtures of the two types of pasta where a preferential insect orientation was not observed due to the presence of both the attractive (Khorasan pasta) and the non-attractive source (chickpea pasta) in both test odour-stimuli.
In the head-space of different pasta samples, a total of 50 volatile compounds were identified by chemical analysis. Even though many of these compounds were found in the head-space fractions of both cereal-and legume-based pastas, differences in their relative proportions along with qualitative differences were highlighted. In fact, the odour profiles of cereal-based pastas mainly contained aldehydes, followed by alcohols, aromatics, furans, and hydrocarbons whereas those of legume-based pastas were characterised by a high content of alcohols, followed by aldehydes, aromatics, furans, and lactones. Short chain aldehydes are lipid oxidation products of the hydroperoxide lyase pathway of oxylipin metabolism [44,45], which can be converted into the corresponding alcohols by the action of alcohol dehydrogenase [46]. Ketones and hydrocarbons are also derived from lipid oxidation, from both enzymatic and non-enzymatic oxidative degradations, while terpenes are naturally present/synthesised by the plant [47]. Lactones are strongly related to legumes [48]. Furans are mainly produced by the Maillard reaction during pasta drying [34,49]. Differences in composition of the pasta volatile fractions might account for differences in attractiveness of various pasta samples to granary weevil adults.
In our study, S. granarius adults strongly preferred Khorasan and, to a lesser extent, five cereal pastas over the other samples tested, with legume-based pastas being less preferred. However, by matching the volatile profiles of pasta samples with the known behavioural activity of some of their components, it is difficult to draw conclusions about the compounds involved in determining the olfactory preference of S. granarius adults. In fact, the volatile fractions of both cereal-and legume-based pastas are comprised of some of the above-mentioned granary weevil attractants and repellents [13], but they represent only a limited number of VOCs, identified in this study, and the behavioural activity of many of these components is still unknown [50]. Therefore, the contributions of other, even minor, volatile compounds emitted by the most attractive pasta samples, alone and in combination with known attractants, deserve further investigation.
The response pattern of insects to different pasta samples did not vary markedly between 1 and 7 days; this strongly suggests that the olfactory preference of insects was mainly determined by the first choice made in response to the odour profiles of different pasta samples. However, since insects remained alive and fed on the pastas during the experiments, the possible release of additional volatiles, mainly by insects, which were not detected in the SPME collections from the pasta samples alone, cannot be excluded. Further studies are needed to confirm differences in the attractiveness of pasta samples to granary weevils in short-term behavioural bioassays and to characterize the SMPE volatile profiles of pastas fed upon by insects to better understand the chemical bases of attraction.
As reported, in susceptibility tests, 100% mortality of adult weevils was recorded 20 days after their exposure to all legume-based pastas and no progeny were recorded during the eight consecutive weeks. Therefore, consistently with results of behavioural bioassays, susceptibility tests demonstrated the unsuitability of legume-based pastas as a food source for adult granary weevils. This is in fairly good agreement with previous studies reporting a significant reduction of survival and reproduction of conspecific S. oryzae adults fed with an admixture of yellow split-peas and wheat [30,31] or rice treated with 1% pea flour extract [51]. Similar results were obtained by Fields et al. (2001) [25] with nine stored-grain beetles, including S. oryzae, S. granarius, and S. zeamais, reared on wheat kernels or flour treated with P. sativum fractions. Altogether, Sitophilus spp. were the most sensitive species and the protein-rich pea fraction was more toxic than the fibre fraction, which was more toxic than the starch fraction [26].
The toxicity of pea albumin 1b (PA1b), a 37 amino-acid peptide extracted from pea seeds, for cereal weevils (Sitophilus spp.) was discovered by Delobe et al. (1998) [33], and a high-affinity binding site for this entomotoxin in susceptible Sitophilus strains was characterised [52]. However, the diverse biological activities of legume fractions towards stored-product pests strongly suggest that the contents of different toxic and deterrent allelochemicals in legume flour remain active, even after the pasta production process, as demonstrated by our study.

Conclusions
From a practical perspective, it is interesting to define the biological activity of volatile and non-volatile components of legume-based pastas, as they could allow the identification of possible repellent, deterrent, and toxic compounds to be used, for example, for the preparation of bioactive packaging able to limit the risk of infestation of packaged products [21,50]. However, more work is needed to determine the linkage between the bioassays and the volatile compounds from the chromatography studies.